Fine-Tuning the Linear Release Rate of Paclitaxel-Bearing Supramolecular Filament Hydrogels through Molecular Engineering Rami W. Chakroun,† Feihu Wang,† Ran Lin,† Yin Wang,† Hao Su,† Danielle Pompa,‡ and Honggang Cui*,†,§,⊥ Downloaded via IDAHO STATE UNIV on July 17, 2019 at 07:45:40 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Department of Chemical and Biomolecular Engineering, and Institute for NanoBiotechnology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States ‡ Department of Biomedical Engineering, University of Utah, 201 Presidents Circle, Salt Lake City, Utah 84112, United States § Department of Oncology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States ⊥ Center for Nanomedicine, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 North Broadway, Baltimore, Maryland 21231, United States S Supporting Information *
ABSTRACT: One key design feature in the development of any local drug delivery system is the controlled release of therapeutic agents over a certain period of time. In this context, we report the characteristic feature of a supramolecular filament hydrogel system that enables a linear and sustainable drug release over the period of several months. Through covalent linkage with a short peptide sequence, we are able to convert an anticancer drug, paclitaxel (PTX), to a class of prodrug hydrogelators with varying critical gelation concentrations. These self-assembling PTX prodrugs associate into filamentous nanostructures in aqueous conditions and consequently percolate into a supramolecular filament network in the presence of appropriate counterions. The intriguing linear drug release profile is rooted in the supramolecular nature of the self-assembling filaments which maintain a constant monomer concentration at the gelation conditions. We found that molecular engineering of the prodrug design, such as varying the number of oppositely charged amino acids or through the incorporation of hydrophobic segments, allows for the fine-tuning of the PTX linear release rate. In cell studies, these PTX prodrugs can exert effective cytotoxicity against glioblastoma cell lines and also primary brain cancer cells derived from patients and show enhanced tumor penetration in a cancer spheroid model. We believe this drugbearing hydrogel platform offers an exciting opportunity for the local treatment of human diseases. KEYWORDS: molecular assembly, drug delivery, controlled release, prodrug, hydrogels, chemotherapy
S
have been reported to self-organize into a wide variety of shapes such as ribbons,32−34 filaments,35−38 and spheres.39,40 From the perspective of molecular engineering, peptide amphiphiles (PAs) are a class of self-assembling peptide conjugates
upramolecular biomaterials have found use in a wide range of biomedical applications such as tissue engineering,1 regenerative medicine,2−5 drug delivery,6−13 molecular imaging,14,15 and immune engineering.16−21 This has led to great advancements in the molecular and supramolecular engineering of these materials on the basis of natural biopolymers,22,23 synthetic polymers,24−26 and peptide-based molecules.5,8,27−31 In particular, peptide-based building units © XXXX American Chemical Society
Received: March 1, 2019 Accepted: May 17, 2019
A
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
www.acsnano.org
Cite This: ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 1. (A) Molecular design of the four studied drug amphiphiles, each containing five key design elements: (i) the paclitaxel drug (red), (ii) a reducible disulfylbutyrate (buSS) linker (green), (iii) a two glycine spacer (blue), (iv) a β-sheet-forming segment consisting of three valines (orange), and (v) a tumor-penetrating (RGDR) sequence (black). (B) Upon dissolution in water, each of the four drug amphiphiles selfassembled into supramolecular filaments which can reversibly dissociate into monomeric DAs upon dilution.
bearing amphiphiles that have been shown to spontaneously associate into discrete supramolecular nanostructures upon dissolution and incubation in aqueous environments.51,60 Under appropriate conditions, the filamentous nanostructures formed by these drug amphiphiles can percolate into a supramolecular network, forming drug-based hydrogels for potential local treatment of diseases. In the context of cancer chemotherapy, localized treatment applies a large amount of therapeutic agents directly at the target site, overcoming the issue exhibited by systemic treatment of substantial drug diffusion into undesired areas of the body. Many systems for localized treatment have been under study, ranging from microdevices61−63 to functionalized nanoparticles,64−68 to hydrogels,69−71 and local administration methods such as convection enhanced delivery.72−74 Gliadel Wafer loaded with carmustine (BCNU) is the only FDA-approved system currently in clinical use for the local treatment of brain tumors.75 This wafer technology consists of BCNU dispersed
containing a hydrophilic peptide sequence covalently linked to a hydrophobic tail (usually an alkyl chain).41,42 Through manipulation of the peptide sequences and assembly conditions, these molecules have been observed to self-assemble into a variety of nanostructures in water41 and demonstrated potential applications in drug delivery,43−46 regenerative medicine,47,48 magnetic resonance imaging (MRI),10,11 and blood vessel growth promotion.49,50 The prodigious self-assembly capabilities of peptide amphiphiles have inspired the design of peptide-based drug conjugates, namely, drug amphiphiles (DAs), where hydrophobic drugs, instead of the alkyl chain, are used to provide the associative, hydrophobic interactions in aqueous solution.51,52 This design strategy has been successfully applied to a number of therapeutic agents such as camptothecin,53−55 paclitaxel,56,57 doxorubicin,58 and others.59 Conjugation of a hydrophilic peptide onto a drug molecule improves its water solubility and, more importantly, constructs a class of self-assembling drugB
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 2. TEM micrographs of low and high magnification for each of the four molecules studied: (A,E) RCP-1, (B,F) RCP-2, (C,G) RCP-3, (D,H) RCP-4. All molecules were aged for 1 week at 1 mM concentration. Insets: respective photos of the hydrogels formed by each of the four molecules are shown above.
(RGDR), a valine−valine−valine segment (VVV), and a double glycine spacer (GG). PTX was chosen to serve as the basis for the drug amphiphile design due to its high efficacy and potency and is known to cause mitotic arrest by promoting microtubule polymerization and stabilization, leading to cell apoptosis.89 In the PTX prodrug design, the free PTX form is expected to recover after the conjugate enters a cell, where elevated glutathione (GSH) concentrations reduce the disulfide linker.57,90 The chosen RGDR peptide sequence has an increased binding affinity to neuropilin-1 (NRP-1), generating an enhanced tissue penetration effect.91 The purpose of including the VVV segment was to promote intermolecular hydrogel bonding among the conjugates. After several failed attempts, we realized that using a GG spacer, instead of a G spacer, to separate the hydrophobic PTX from the peptide segment, helps improve DA solubility as well as enhance the filament flexibility, producing a more robust gel (Figure S5). RCP-1 and RCP-2 incorporated two repeats and a single repeat of the oppositely charged arginine−aspartic acid pair, respectively, whereas RCP3 did not include the oppositely charged two amino acid sequence and RCP-4 incorporated a dodecyl chain to increase hydrophobicity. The purpose of incorporating the oppositely charged amino acids was to improve water solubility while maintaining the overall net charge density of these molecules. All the drug amphiphiles were synthesized using standard Fmoc solid-phase peptide synthesis protocols to produce each peptide, followed by conducting a separate disulfide formation reaction between PTX−buSS−pyridine and the cysteine amino acid of the peptide sequence. The molecules were then purified using high-performance liquid chromatography (HPLC) (Figure S2). Upon being aged in water for several days (typically a week), these DAs were found to form long and flexible supramolecular filaments of several micrometers in length (Figure 1B). Molecular Self-Assembly and Characterization. To understand the gelation properties of each molecule, we sought a deeper understanding of their self-assembly behavior and structural morphology. In these experiments, all four molecules were aged in water at 1 mM concentration at room temperature for 1 week to ensure that the observed assemblies reach a relatively more stable state, although filamentous nanostructures could be observed to appear after 1 day incubation (Figure S3).
within polifeprosan 20 polymer, which degrades naturally over time to release the loaded drug.76 As water influx is restricted to the surface of the wafers, consecutive surface layers degrade, ensuring the drug is released steadily over time, instead of being released in bulk, minimizing system toxicity.77,78 However, the change in surface area during erosion alters the amount of drug released at each stage, resulting in an overall nonlinear profile lasting a few days.79−81 Factors that change erosion behavior and thereby control the rate of release range from polymer biodegradability,82 to molecular weight,83 to composition.84,85 Another FDA-approved biodegradable material that is extensively used in clinic is poly(lactic-co-glycolic acid) (PLGA). Its biodegradable nature and controllable degradation, through polymer composition and molecular weight, have made it a preferred candidate for drug delivery.86 However, such material often exhibits a nonlinear release profile (mainly burst release), limiting its use as an efficient drug carrier.87,88 Given the aggressive nature of many tumors, a system that provides longterm, sustainable, and controlled drug release is necessary to achieve improved treatment outcomes. In this context, we report our design of self-assembling paclitaxel (PTX) drug amphiphiles to precisely regulate the rate of drug release in a dissociation-controlled manner. Our rationally crafted PTX-peptide conjugates can spontaneously associate into injectable supramolecular hydrogels, which linearly release the monomeric conjugates via network disruption and molecular dissolution. Through molecular engineering, we are able to tune the instability of these drugbased hydrogels, enabling the accurate modulation of the drug release rate.
RESULTS AND DISCUSSION Molecular Design. Four variations of PTX prodrug hydrogelators were designed with the purpose of modulating the release kinetics of supramolecular hydrogels through the incorporation of oppositely charged amino acids and an alkyl chain onto the peptidic segment. As shown in Figure 1A, all four molecules shared some common design features: a reducible biodegradable linker, 4-(pyridin-2-yl-disulfanyl)butyrate (buSS), conjugating PTX to a peptide sequence consisting of an arginine−glycine−aspartic acid−arginine sequence C
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
PTX absorption,95 acting in concert with different molecular twisting imposed by the bulky PTX. The difference in strength of the β-sheet signals is believed to correspond to the alternation in internal molecular stacking and the population of filaments formed as a result of their different CAC values. In contrast, RCP-1 showed a negative peak at 206 nm, indicative of a random coil secondary structure as the dominant conformation. This observation was consistent with the TEM imaging (Figure 2A), which displays a large number of spherical assemblies. In the literature, separate studies by Shimada et al.96 and Lock et al.97 reported the presence of spherical assemblies as a precursor morphology prior to formation of filamentous nanostructures and correlated the presence of spherical assemblies to the random coil secondary structure, whereas the emergence of filamentous structures is consequential to the directional, intermolecular hydrogen bonding.98 We next studied the gelation and aggregation potentials of the four molecules. Critical gelation concentration (CGC) represents the minimum concentration of conjugates required to form a self-supporting hydrogel. In order to obtain the CGC value, these conjugates were aged at varying concentrations for 2 days at room temperature, then PBS was added to stimulate the gelation. Using a simple inversion test, the CGC of RCP-1 was determined to be between 2 and 4 mM; CGC values for RCP-2 and RCP-3 were between 0.5 and 1 mM, and that of RCP-4 was below 0.5 mM (Figure S6). The difference in CGC values is attributed to the varying molecular design that led to subtle yet discernible change in their assembly behavior. The critical aggregation concentration (CAC) of each molecule was further determined using a Nile Red encapsulation assay. As illustrated in Figure 3B, Nile Red in solution form displays a peak intensity of ∼660 nm, which shifts to 635 nm when it is encapsulated within a hydrophobic environment.99 Drug conjugates were dissolved and aged at different concentrations in the presence of Nile Red and then run through a fluorimeter to measure the fluorescence intensity at different wavelengths. As presented in Figure 3C and calculated using the intensity ratio (Figure S4), RCP-1 exhibited the highest CAC value of 8.2 μM, with that of RCP-2 at 3.7 μM, RCP-3 at 2.1 μM, and RCP-4 with the lowest value of 0.11 μM. This trend in CAC values directly correlates with the intensity of the β-sheet absorption in CD and the selfassembling structures revealed by TEM. These findings suggest that increasing the number of charged amino acids within the DAs results in formation of filaments with higher CAC values. At the same time, incorporating an alkyl chain into the molecular design greatly reduced the CAC value as a result of enhanced hydrophobic interactions. Drug Release Kinetics. CAC values are indicative of conjugates’ tendency to form assemblies above a threshold concentration and also provide information on the dissociation potential of filaments into free conjugates upon dilution. In our filament hydrogel system, we speculate that the PTX release rate is primarily limited by the rate of filament dissociation (Figure 4A), which is linked to the respective CAC value. To verify this, we performed release studies on the hydrogels formed by the four conjugates. A fixed volume of PBS solution was placed on top of each hydrogel to act as a release medium, which was removed and replaced with an equal volume of fresh PBS solution at predetermined time intervals. The collected medium was analyzed using HPLC to determine the exact concentration of the released drug conjugate with a calibration curve. Please note that we are assessing the release rate of the monomeric conjugate, not the free PTX. The liberation of free PTX from the
Figure 2 demonstrates the representative transmission electron microscopy (TEM) images of the four molecules under study. Low-magnification images (Figure 2A−D) revealed all four molecules self-assembled into filamentous nanostructures. RCP1 showed the tendency to self-assemble into filaments but coexist with many spherical structures at 1 mM (Figure 2A). Upon further magnification, we found that these filaments display a ribbon-like morphology with slightly different forms of twisting. A clear transition in morphology was observed with an increasing number of oppositely charged amino acids (RCP-1 to RCP-3: Figure 2E−G). Such structural twisting is a common phenomenon seen in many peptide assembly systems, resulting from the inherent chirality of natural amino acids and the difference in internal packing characteristics rooted in each molecular design.92−94 Upon addition of phosphate-buffered saline (PBS) solution to the conjugate solutions (a final 1× salt concentration was reached), RCP-2, RCP-3, and RCP-4 all formed strong hydrogels, whereas RCP-1 formed large agglomerates in solution at the concentration studied (no gelation behavior was observed unless the concentration was increased higher than 2 mM; see insets in Figure 2). To further understand their self-assembly behavior, we used circular dichroism (CD) to probe the molecular packing within the observed supramolecular filaments. Figure 3A shows the CD spectra of all four molecules. From these results, it can be said that RCP-2, RCP-3, and RCP-4 all formed filaments rich in βsheets, as evidenced by the negative peak around 220 nm. The slight red shift observed could be a result of overlapping with the
Figure 3. (A) Circular dichroism spectra for all four molecules in water. (B) Schematic illustration indicating the use of Nile Red as an encapsulating agent to determine the CAC value of self-assembled filaments. (C) Fluorescence measurements used to determine the CAC value using the Nile Red encapsulation method. D
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
phase of release comes into effect: all filaments start to dissociate into monomers until a new equilibrium is established (Figure 5B). As network disruption and filament dissociation only happens at the interface, the gel shrunk downward over time. Therefore, the surface area of contact between the hydrogel and the surrounding fluid would play a crucial role in determining the release behavior. To further validate our release mechanism, a second release experiment was conducted with RCP-3 gels formed at three different concentrations: 1, 2, and 3 mM. RCP-3 gel was chosen for this study as it presents as a clear and sturdy gel, making it easier to observe water influx and network disruption. Data points were collected every 2 days for 2 weeks. The hydrogels aged at a higher concentration exhibited a larger accumulation of drug conjugate in the release medium (Figure S7), yet they displayed a slower release by percentage (it actually released more drug in terms of concentration) (Figure 5E). This is well expected because the more concentrated gel showed greater resistance to gel swelling and disruption (Figures 5D and S7). The presence of short filaments explains the concentrationdependent release behavior and that the PTX concentration of the release media sometimes is higher than the actual CAC value of each respective DA. The 3 mM gel contains a larger population of filaments in contact with the water during the dissociation phase, causing more filaments to diffuse into the released medium. In Vitro Cytotoxicity. In order to assess the effect of peptide conjugation on drug efficacy, two in vitro experiments were carried out: disulfide bond cleavage to release free PTX and cytotoxicity assays to assess the drug conjugate’s efficacy. Glutathione, a disulfide bond reducer found at 10 mM concentrations in the cytosol, is known to reduce the disulfide bond and release free PTX with time.90 To mimic the typical conditions inside cells, drug conjugate was dissolved in water at a 50 μM concentration and incubated at 37 °C for 100 h, both in the presence and in the absence of 10 mM glutathione. At certain time points, a fixed portion of the solution was withdrawn and analyzed by HPLC to determine the remaining conjugate concentration. Our results (Figure 6A) show that drug conjugate reduction took place very effectively, with the majority of the free PTX released within the first day. In the absence of GSH, the drug conjugate was very stable, with less than 20% degradation observed over 100 h. The degradation without GSH is attributable to hydrolysis that breaks the ester bond between free PTX and the adjacent spacer. The cytotoxic activity of the PTX prodrug was assessed on the U87 glioblastoma cell line and the 612 human primary brain tumor cells. As a measure of cytotoxicity, an IC50 study was conducted using a MTT and AlamarBlue assay. As shown in Figure 6B,C, there was no substantial reduction in cytotoxicity for all of the designed conjugates. RCP-1 had IC50 values of 103 and 32 nM; RCP-2 had IC50 values of 48 and 14 nM; RCP-3 had IC50 values of 36 and 8.6 nM; RCP-4 had IC50 values of 220 and 69 nM, whereas free PTX had IC50 values of 14 and 1.7 nM on U87 and 612 cell lines, respectively. At the same time, the negligible cytotoxicity observed from the peptide alone (Table S1) implies the biocompatibility of the chosen peptide. Given the efficient conversion of PTX conjugate to free PTX within cells, there are two major factors contributing to the observed toxicity: filament dissociation into conjugate monomers and the consequent cellular uptake. Formation of long, stable filament reduces its cellular uptake efficiency and also slows down the dissociation kinetics into short filaments and monomeric
Figure 4. (A) Illustration showing an overview of the gel release process from the supramolecular hydrogels, with the rate of filament disassembly governing the overall release kinetics. (B) Gel release profile of all four molecules taken at equal time intervals over a 1 month period. Hydrogels were incubated at 37 °C. Data are given as mean ± SD (n = 3).
conjugate was studied in the presence of glutathione (vide infra). As expected, RCP-1, having the highest CAC value, showed the fastest release rate, whereas RCP-4, having the lowest CAC value, showed the slowest release (Figure 4B). Very importantly, a linear release of drug conjugate was observed for all four hydrogels. Notably, the release of only ∼1−7% of the conjugate over the period of 1 month gives a good prediction of their longterm release characteristics. Proposed Mechanism of Release. We speculate that the release of PTX conjugates occurs primarily at the interface between the supramolecular hydrogel and PBS solution (Figure 5A). When PBS solution is placed on top of the gel, supramolecular filaments are expected to dissociate and diffuse into the PBS solution, accompanied by water penetration into the hydrogels. Indeed, TEM imaging of the release media revealed the presence of short filaments and some spherical assemblies of RCP-3 (Figure 5B), indicating that not only free PTX conjugate but smaller assemblies can also be released out. Interestingly, instead of gradual swelling or overall size reduction, the gel was seen to shrink downward during the release period, as evidenced by a gradual drop of the interface level (Figure 5C). In consideration of all the observations, a twophase release mechanism was proposed: the first phase involves local gel swelling/network disruption at the interface, immediately after the PBS solution addition. Due to a difference in osmolarity, monomeric conjugates started to diffuse into the PBS solution, while water in the release medium penetrates into the filament network, causing the filaments to spread out and to disrupt the network. As the conjugate concentration within the surrounding medium drops below the CAC value, the second E
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 5. (A) Schematic illustration of the proposed two-step release mechanism: local gel swelling/network disruption followed by filament dissociation. (B) TEM image of a collected release sample proving the diffusion of filament fragments out of the gel into the surrounding fluid. (C) Images of the gel condition at different time points throughout the release experiment, showing a downward breakdown of the gel. (D) Images of the 3, 2, and 1 mM RCP-3 gel samples 10 days into the release experiment. As indicated by the arrows, less concentrated gels showed larger and deeper areas of water influx. (E) RCP-3 gel release profiles conducted at three different concentrations (1, 2, and 3 mM) with release samples collected at equal time intervals. Data are given as mean ± SD (n = 3).
Figure 6. (A) Paclitaxel release study in PBS buffer at 37 °C at a drug conjugate concentration of 50 μM. Solutions were incubated in the presence or absence of 10 mM GSH. Cytotoxicity evaluation of all four molecules and free PTX against U87 glioblastoma cells (B) and 612 human primary brain tumor cells (C). Cells were incubated with the PTX or conjugates for 72 h, and cell viability was determined by MTT assay (U87 cell line) and AlamarBlue assay (612 cell line). IC50 values are provided in the bar chart legends. Data are given as mean ± SD (n = 3).
conjugates at 10 μM concentration to the spheroids, the inhibitory effect was evaluated by measuring volume change of these spheroids compared to the first day of treatment (day 0). Although free PTX is slightly more toxic than the four conjugates, Figure 7A,B shows that our drug conjugates demonstrate greater tumor inhibition in the 3D spheroid model. Over 2 weeks, free PTX reduced the tumor spheroid volume to 81% of the original size, whereas RCP-1, RCP-2, RCP-3, and RCP-4 reduced it to 77, 67, 61, and 80%, respectively. The control group showed a 530% increase in tumor size. The change in background color reflected media discoloration as a result of pH change due to cell proliferation.
conjugates. Therefore, it is not surprising to see that RCP-4, the PTX prodrug with the lowest CAC value, shows the highest IC50 to both types of cells. On the other hand, RCP-1 might be too water-soluble, having a low Log P value that impedes its ability to passively diffuse across the cell membranes, thus possessing a relatively high IC50 value. Inhibition and Penetration in a Tumor Spheroid Model. The RGDR sequence was incorporated at the peptide termini with the goal of improving its ability to penetrate into the tumor.100 To assess this effect, U87 cancer cells were cultured into tumor spheroids using agarose gel-coated plates. An inhibition study was conducted by adding free PTX and drug F
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
Figure 7. Assessment of PTX supramolecular filament hydrogels in a spheroid model. (A) Photos showing tumor spheroid growth following treatment with free PTX and all four DAs at a concentration of 10 μM. Spheroids were treated with drug-free Dulbecco’s modified Eagle’s medium to serve as the control group. (B) Graph showing the relative volume of the tumor spheroids compared with day 0. Data are given as mean ± SD (n = 4). (C) Confocal laser scanning microscopy (CLSM) images of a U87 tumor spheroid showing the uptake of 5FAM-labeled RGDR peptide and free 5FAM at different cross sections within the tumor after 48 h incubation time. (D) Analysis of the CLSM images quantifying cellular uptake of the 5FAM peptide conjugate by U87 cells. Flow cytometry was used to measure the percentage of fluorescent cells, denoted by M2, for 0 h (i), 24 h (ii), and 48 h incubation (iii), and to measure the fluorescence intensity of cells after 24 and 48 h incubation times (iv).
To confirm that the enhancement in tumor inhibition of these four conjugates is a result of the RGDR sequence, a tumor penetration study was conducted by incubating U87 tumor spheroids with a 5FAM−RGDR conjugate. Images from a confocal laser scanning microscope and flow cytometry data were obtained to provide both a qualitative and quantitative analysis of the penetration effect. Compared to control molecule 5FAM, 5FAM−RGDR showed enhanced penetration into the tumor, reaching areas up to 150 μm in depth (Figure 7C). Flow cytometry data of the cells within the spheroid showed that at 24 h incubation time, 40% of the cells displayed a right positive fluorescence shift for 5FAM−RGDR (Figure 7D,ii), and at 48 h, 53.8% of the cells gave a positive fluorescence shift (Figure 7D,iii), in comparison to the control group (Figure 7D,i). Fluorescence intensity measurements confirm that incubating cells for a longer period of time results in greater cellular uptake of conjugate (Figure 7D,iv). These results indicate that the tumor penetration effect of the RGDR sequence contributes to the enhanced cytotoxicity of the drug conjugates within the spheroid model.
release observed in many drug delivery systems, is rooted in the supramolecular nature of the reported system capable of maintaining a constant monomer concentration under the gelation conditions. The exact release rate is dependent on both the networking property of the gel at the interface and the instability of the filaments in bulk. From the perspectives of molecular engineering, incorporating an alkyl chain lowers the CAC value, resulting in a much slower release profile. Increasing the number of oppositely charged amino acids in the peptidic segment led to a higher CAC value, subsequently accelerating the dissociation kinetics of the supramolecular filaments into monomers. In all cases, the self-assembling prodrug system exhibits a long-term linear release profile. Furthermore, we demonstrated that conjugating a RGDR-bearing peptide on the anticancer drug paclitaxel not only preserves cytotoxic effects but also promotes tumor inhibition in a spheroid model. We believe that the linear release characteristics and tunability displayed by our PTX-bearing hydrogel system enable their great potential to improve local treatment of cancer and other human diseases.
CONCLUSION The work reported in this study demonstrated the characteristic drug release feature of drug-bearing supramolecular filament hydrogels and our ability to fine-tune the release rate of PTX prodrugs from their hydrogel form through molecular engineering. The linear release profile, in sharp contrast to the burst
MATERIALS AND METHODS Molecular Self-Assembly and Transmission Electron Microscopy. All four drug conjugates were aged for a week in water at a 1 mM concentration at room temperature, unless further specified. Dilutions to 100 μM were made right before preparing the TEM grids. Ten microliters of sample solution was deposited onto a carbon-coated copper grid (Electron Microscopy Services, Hatfield, PA). After 1 min, G
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano the excess solution was wicked away using filter paper. Immediately after, 10 μL of a 2 wt % aqueous uranyl acetate solution was deposited on the grid for 30 s before the excess solution was removed. The sample grid was left to dry at room temperature for 3 h prior to imaging. Brightfield TEM imaging was performed on a FEI Tecnai 12 TWIN transmission electron microscope operated at an acceleration voltage of 100 kV. All TEM images were recorded by a SIS Megaview III wideangle CCD camera or 16 bit 2K × 2K FEI Eagle bottom mount camera.53,57 Drug Release Studies. Drug conjugates were aged at a 1 mM concentration for 2 days at room temperature. Next, 200 μL of each solution was then aliquoted into three 500 μL vials. Twenty microliters of 10× PBS was added to each solution to stimulate gel formation. Then, 75 μL of 1× PBS was then added to the surface of each gel. The vials were incubated at 37 °C in a water bath. At each time point, 50 μL of PBS was removed and replaced with 50 μL of fresh 1× PBS. The conjugate concentration of release medium samples was determined via analytical HPLC using a previously derived calibration curve based on the area under the corresponding peak. Degradation Study. The release characteristics of RCP-2 were studied at 100 μM concentration. A 20 mM glutathione stock solution was prepared in 2× PBS buffer (pH 7.4), and solutions of the conjugate (200 μM) were prepared in water and aged for 2 days. Equal volumes of conjugate and GSH solution were then mixed to reach a final GSH concentration of 10 mM and a final conjugate concentration of 100 μM. Three vials of the solution mixture were incubated at 37 °C for a total of 100 h. Samples were taken at each time point, and release of paclitaxel from conjugate was detected by HPLC. The 2× PBS buffer was used to prepare the negative control that contained no GSH.57 Cell Viability Study. A dose−response study was used to evaluate the cytotoxicity of each drug conjugate. A 96-well plate was seeded with a cell density of 5000 cells/well and incubated at 37 °C for 24 h. The cells were then treated with varying concentrations of paclitaxel or conjugate in cell medium and incubated for a further 72 h. Cell viability was determined by MTT (U87 cell line) and AlamarBlue (612 cell line) assays (Sigma-Aldrich) according to the manufacturer’s protocol. Tumor Inhibition and Penetration Assay. A 24-well plate was base-coated with 2% agarose. U87 cells were seeded onto the plate with a cell density of 10 000 cells/well and incubated at 37 °C. After 24 h, the cells migrated to the center of the wells and formed tumor spheroids. To carry out the tumor inhibition study, the spheroids were left to grow for 7 days. After such, the cell medium was removed, replaced and a drug conjugate solution, and then added to reach a final concentration of 10 μM. At each time point (0, 2, 4, 6, 8, 12, 14 days), the spheroids were imaged under an inverted microscope (EVOS XL Core) and the diameter was measured manually. To carry out the tumor penetration study, tumor spheroids were treated with 5FAM−RGDR and 5FAM (5 μM) for 48 h. After incubation, tumor spheroids were washed four times with ice cold PBS, fixed with formaldehyde (10% w/v in PBS) for 30 min, and placed in cavity microscope slides. Images of the spheroid centers were acquired by tomoscan using confocal laser scanning microscopy (Zeiss, LSM510, Germany). For quantitative determination of penetration efficiency, the U87 MG glioma spheroids were incubated with 5FAM−RGDR for 12 and 24 h. Subsequently, the glioma spheroids were washed four times with ice cold PBS and lysed with 0.5% Triton-X. The percentage of 5FAM-positive cells and fluorescence intensity was detected using a flow cytometer (FACS Calibur, BD).
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Feihu Wang: 0000-0002-0358-967X Ran Lin: 0000-0002-4413-6300 Honggang Cui: 0000-0002-4684-2655 Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS The work reported here is supported by the National Science Foundation (DMR 1255281), and R.W.C. acknowledges the support of NSF Graduate Research Fellowships Program (DGE 1746891). We thank the Integrated Imaging Center (IIC) at The Johns Hopkins University for TEM imaging. We also thank Dr. Alfredo Quiñones-Hinojosa (Department of Neurosurgery, Mayo Clinic) for providing the primary brain tumor cells. REFERENCES (1) Clarke, D. E.; Pashuck, E. T.; Bertazzo, S.; Weaver, J. V.; Stevens, M. M. Self-Healing, Self-Assembled β-Sheet Peptide-Poly (L-Glutamic Acid) Hybrid Hydrogels. J. Am. Chem. Soc. 2017, 139, 7250−7255. (2) Hou, S.; Wang, X.; Park, S.; Jin, X.; Ma, P. X. Rapid Self Integrating, Injectable Hydrogel for Tissue Complex Regeneration. Adv. Healthcare Mater. 2015, 4, 1491−1495. (3) Angeloni, N. L.; Bond, C. W.; Tang, Y.; Harrington, D. A.; Zhang, S.; Stupp, S. I.; McKenna, K. E.; Podlasek, C. A. Regeneration of the Cavernous Nerve by Sonic Hedgehog Using Aligned Peptide Amphiphile Nanofibers. Biomaterials 2011, 32, 1091−1101. (4) Tysseling-Mattiace, V. M.; Sahni, V.; Niece, K. L.; Birch, D.; Czeisler, C.; Fehlings, M. G.; Stupp, S. I.; Kessler, J. A. Self-Assembling Nanofibers Inhibit Glial Scar Formation and Promote Axon Elongation after Spinal Cord Injury. J. Neurosci. 2008, 28, 3814−3823. (5) Ellis-Behnke, R. G.; Liang, Y.-X.; You, S.-W.; Tay, D. K.; Zhang, S.; So, K.-F.; Schneider, G. E. Nano Neuro Knitting: Peptide Nanofiber Scaffold for Brain Repair and Axon Regeneration with Functional Return of Vision. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5054−5059. (6) Kuang, Y.; Du, X.; Zhou, J.; Xu, B. Supramolecular Nanofibrils Inhibit Cancer Progression In Vitro and In Vivo. Adv. Healthcare Mater. 2014, 3, 1217−1221. (7) Soukasene, S.; Toft, D. J.; Moyer, T. J.; Lu, H.; Lee, H.-K.; Standley, S. M.; Cryns, V. L.; Stupp, S. I. Antitumor Activity of Peptide Amphiphile Nanofiber-Encapsulated Camptothecin. ACS Nano 2011, 5, 9113−9121. (8) Altunbas, A.; Lee, S. J.; Rajasekaran, S. A.; Schneider, J. P.; Pochan, D. J. Encapsulation of Curcumin in Self-Assembling Peptide Hydrogels as Injectable Drug Delivery Vehicles. Biomaterials 2011, 32, 5906− 5914. (9) Mulyasasmita, W.; Cai, L.; Hori, Y.; Heilshorn, S. C. AvidityControlled Delivery of Angiogenic Peptides from Injectable MolecularRecognition Hydrogels. Tissue Eng., Part A 2014, 20, 2102−2114. (10) Webber, M. J.; Matson, J. B.; Tamboli, V. K.; Stupp, S. I. Controlled Release of Dexamethasone from Peptide Nanofiber Gels to Modulate Inflammatory Response. Biomaterials 2012, 33, 6823−6832. (11) Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165−13307. (12) Li, Y.; Wang, F.; Cui, H. Peptide Based Supramolecular Hydrogels for Delivery of Biologics. Bioeng. Transl. Med. 2016, 1, 306− 322. (13) Monroe, M.; Flexner, C.; Cui, H. Harnessing Nanostructured Systems for Improved Treatment and Prevention of HIV Disease. Bioeng. Transl. Med. 2018, 3, 102−123.
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01689. Details of molecular characterization, additional TEM images of the assembled supramolecular structures by the designed PTX prodrugs, fluorescence spectrometry for CAC measurement, gel release and cell cytotoxicity study (PDF) H
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils. ACS Nano 2017, 11, 8579−8589. (35) Pashuck, E. T.; Duchet, B. J.; Hansel, C. S.; Maynard, S. A.; Chow, L. W.; Stevens, M. M. Controlled Sub-Nanometer Epitope Spacing in a Three-Dimensional Self-Assembled Peptide Hydrogel. ACS Nano 2016, 10, 11096−11104. (36) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Spontaneous Assembly of a Self-Complementary Oligopeptide to Form a Stable Macroscopic Membrane. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 3334− 3338. (37) Zhang, S. Fabrication of Novel Biomaterials through Molecular Self-Assembly. Nat. Biotechnol. 2003, 21, 1171−1178. (38) Moreira, I. S. P.; Piskorz, T. K.; van Esch, J. H.; Tuttle, T.; Ulijn, R. V. Biocatalytic Self-Assembly of Tripeptide Gels and Emulsions. Langmuir 2017, 33, 4986−4995. (39) Pashuck, E. T.; Cui, H.; Stupp, S. I. Tuning Supramolecular Rigidity of Peptide Fibers through Molecular Structure. J. Am. Chem. Soc. 2010, 132, 6041. (40) Fields, G. B.; Lauer, J. L.; Dori, Y.; Forns, P.; Yu, Y. C.; Tirrell, M. Proteinlike Molecular Architecture: Biomaterial Applications for Inducing Cellular Receptor Binding and Signal Transduction. Biopolymers 1998, 47, 143−151. (41) Cui, H.; Webber, M. J.; Stupp, S. I. Self Assembly of Peptide Amphiphiles: From Molecules to Nanostructures to Biomaterials. Biopolymers 2010, 94, 1−18. (42) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Peptide-Amphiphile Nanofibers: A Versatile Scaffold for the Preparation of Self-Assembling Materials. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5133−5138. (43) Guler, M. O.; Claussen, R. C.; Stupp, S. I. Encapsulation of Pyrene within Self-Assembled Peptide Amphiphile Nanofibers. J. Mater. Chem. 2005, 15, 4507−4512. (44) Zhang, P.; Cheetham, A. G.; Lin, Y.-A.; Cui, H. Self-Assembled Tat Nanofibers as Effective Drug Carrier and Transporter. ACS Nano 2013, 7, 5965. (45) Soukasene, S.; Toft, D. J.; Moyer, T. J.; Lu, H.; Lee, H.-K.; Standley, S. M.; Cryns, V. L.; Stupp, S. I. Anti-Tumor Activity of Peptide Amphiphile Nanofiber-Encapsulated Camptothecin. ACS Nano 2011, 5, 9113. (46) Accardo, A.; Tesauro, D.; Mangiapia, G.; Pedone, C.; Morelli, G. Nanostructures by Self Assembling Peptide Amphiphile as Potential Selective Drug Carriers. Biopolymers 2007, 88, 115−121. (47) Beniash, E.; Hartgerink, J. D.; Storrie, H.; Stendahl, J. C.; Stupp, S. I. Self-Assembling Peptide Amphiphile Nanofiber Matrices for Cell Entrapment. Acta Biomater. 2005, 1, 387−397. (48) Matson, J. B.; Stupp, S. I. Self-Assembling Peptide Scaffolds for Regenerative Medicine. Chem. Commun. 2012, 48, 26−33. (49) Rajangam, K.; Behanna, H. A.; Hui, M. J.; Han, X.; Hulvat, J. F.; Lomasney, J. W.; Stupp, S. I. Heparin Binding Nanostructures to Promote Growth of Blood Vessels. Nano Lett. 2006, 6, 2086−2090. (50) Rajangam, K.; Arnold, M. S.; Rocco, M. A.; Stupp, S. I. Peptide Amphiphile Nanostructure-Heparin Interactions and Their Relationship to Bioactivity. Biomaterials 2008, 29, 3298−3305. (51) Su, H.; Koo, J. M.; Cui, H. One-Component Nanomedicine. J. Controlled Release 2015, 219, 383−395. (52) Cheetham, A. G.; Chakroun, R. W.; Ma, W.; Cui, H. SelfAssembling Prodrugs. Chem. Soc. Rev. 2017, 46, 6638−6663. (53) Cheetham, A. G.; Zhang, P.; Lin, Y.-A.; Lock, L. L.; Cui, H. Supramolecular Nanostructures Formed by Anticancer Drug Assembly. J. Am. Chem. Soc. 2013, 135, 2907−2910. (54) Cheetham, A. G.; Lin, Y.-a.; Lin, R.; Cui, H. Molecular Design and Synthesis of Self-Assembling Camptothecin Drug Amphiphiles. Acta Pharmacol. Sin. 2017, 38, 874−884. (55) Cheetham, A. G.; Ou, Y.-C.; Zhang, P.; Cui, H. LinkerDetermined Drug Release Mechanism of Free Camptothecin from SelfAssembling Drug Amphiphiles. Chem. Commun. 2014, 50, 6039−6042. (56) Gao, Y.; Kuang, Y.; Guo, Z.-F.; Guo, Z.; Krauss, I. J.; Xu, B. Enzyme-Instructed Molecular Self-Assembly Confers Nanofibers and a Supramolecular Hydrogel of Taxol Derivative. J. Am. Chem. Soc. 2009, 131, 13576−13577.
(14) Bull, S. R.; Guler, M. O.; Bras, R. E.; Meade, T. J.; Stupp, S. I. SelfAssembled Peptide Amphiphile Nanofibers Conjugated to MRI Contrast Agents. Nano Lett. 2005, 5, 1−4. (15) Bull, S. R.; Guler, M. O.; Bras, R. E.; Venkatasubramanian, P. N.; Stupp, S. I.; Meade, T. J. Magnetic Resonance Imaging of SelfAssembled Biomaterial Scaffolds. Bioconjugate Chem. 2005, 16, 1343− 1348. (16) Hudalla, G. A.; Modica, J. A.; Tian, Y. F.; Rudra, J. S.; Chong, A. S.; Sun, T.; Mrksich, M.; Collier, J. H. A Self Adjuvanting Supramolecular Vaccine Carrying a Folded Protein Antigen. Adv. Healthcare Mater. 2013, 2, 1114−1119. (17) Black, M.; Trent, A.; Kostenko, Y.; Lee, J. S.; Olive, C.; Tirrell, M. Self Assembled Peptide Amphiphile Micelles Containing a Cytotoxic T Cell Epitope Promote a Protective Immune Response In Vivo. Adv. Mater. 2012, 24, 3845−3849. (18) Rudra, J. S.; Tian, Y. F.; Jung, J. P.; Collier, J. H. A SelfAssembling Peptide Acting as an Immune Adjuvant. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 622−627. (19) Rudra, J. S.; Sun, T.; Bird, K. C.; Daniels, M. D.; Gasiorowski, J. Z.; Chong, A. S.; Collier, J. H. Modulating Adaptive Immune Responses to Peptide Self-Assemblies. ACS Nano 2012, 6, 1557−1564. (20) Webber, M. J.; Appel, E. A.; Meijer, E.; Langer, R. Supramolecular Biomaterials. Nat. Mater. 2016, 15, 13. (21) Barrett, J. C.; Ulery, B. D.; Trent, A.; Liang, S.; David, N. A.; Tirrell, M. V. Modular Peptide Amphiphile Micelles Improving an Antibody-Mediated Immune Response to Group A Streptococcus. ACS Biomater. Sci. Eng. 2017, 3, 144−152. (22) Daamen, W. F.; Veerkamp, J.; Van Hest, J.; Van Kuppevelt, T. Elastin as a Biomaterial for Tissue Engineering. Biomaterials 2007, 28, 4378−4398. (23) Yamada, Y.; Schneider, J. P. Fragmentation of Injectable Bioadhesive Hydrogels Affords Chemotherapeutic Macromolecules. Biomacromolecules 2016, 17, 2634−2641. (24) Jeong, B.; Bae, Y. H.; Kim, S. W. In Situ Gelation of PEG PLGA PEG Triblock Copolymer Aqueous Solutions and Degradation Thereof. J. Biomed. Mater. Res. 2000, 50, 171−177. (25) Lutolf, M.; Hubbell, J. Synthetic Biomaterials as Instructive Extracellular Microenvironments for Morphogenesis in Tissue Engineering. Nat. Biotechnol. 2005, 23, 47−55. (26) Sionkowska, A. Current Research on the Blends of Natural and Synthetic Polymers as New Biomaterials: Review. Prog. Polym. Sci. 2011, 36, 1254−1276. (27) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. Responsive Hydrogels from the Intramolecular Folding and Self-Assembly of a Designed Peptide. J. Am. Chem. Soc. 2002, 124, 15030−15037. (28) Ulijn, R. V.; Smith, A. M. Designing Peptide Based Nanomaterials. Chem. Soc. Rev. 2008, 37, 664−675. (29) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. PeptideBased Stimuli-Responsive Biomaterials. Soft Matter 2006, 2, 822−835. (30) Li, J.; Gao, Y.; Kuang, Y.; Shi, J.; Du, X.; Zhou, J.; Wang, H.; Yang, Z.; Xu, B. Dephosphorylation of D-Peptide Derivatives to Form Biofunctional, Supramolecular Nanofibers/Hydrogels and Their Potential Applications for Intracellular Imaging and Intratumoral Chemotherapy. J. Am. Chem. Soc. 2013, 135, 9907. (31) Zou, Q. L.; Abbas, M.; Zhao, L. Y.; Li, S. K.; Shen, G. Z.; Yan, X. H. Biological Photothermal Nanodots Based on Self-Assembly of Peptide Porphyrin Conjugates for Antitumor Therapy. J. Am. Chem. Soc. 2017, 139, 1921−1927. (32) Aggeli, A.; Bell, M.; Boden, N.; Keen, J.; et al. Responsive Gels Formed by the Spontaneous Self-Assembly of Peptides into Polymeric Beta-Sheet Tapes. Nature 1997, 386, 259. (33) Deechongkit, S.; Powers, E. T.; You, S.-L.; Kelly, J. W. Controlling the Morphology of Cross β-Sheet Assemblies by Rational Design. J. Am. Chem. Soc. 2005, 127, 8562−8570. (34) Wang, S.-T.; Lin, Y.; Spencer, R. K.; Thomas, M. R.; Nguyen, A. I.; Amdursky, N.; Pashuck, E. T.; Skaalure, S. C.; Song, C. Y.; Parmar, P. A.; et al. Sequence-Dependent Self-Assembly and Structural Diversity I
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano
istration in Treating Intracranial Glioblastoma Xenografts. Neuro. Oncol. 2013, 15, 189−197. (74) Mamot, C.; Nguyen, J. B.; Pourdehnad, M.; Hadaczek, P.; Saito, R.; Bringas, J. R.; Drummond, D. C.; Hong, K.; Kirpotin, D. B.; McKnight, T.; et al. Extensive Distribution of Liposomes in Rodent Brains and Brain Tumors Following Convection-Enhanced Delivery. J. Neuro-Oncol. 2004, 68, 1−9. (75) Chakroun, R. W.; Zhang, P.; Lin, R.; Schiapparelli, P.; Quinones Hinojosa, A.; Cui, H. Nanotherapeutic Systems for Local Treatment of Brain Tumors. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2018, 10, e1479. (76) Dang, W.; Daviau, T.; Brem, H. Morphological Characterization of Polyanhydride Biodegradable Implant Gliadel® During In Vitro and In Vivo Erosion Using Scanning Electron Microscopy. Pharm. Res. 1996, 13, 683−691. (77) Brem, H.; Langer, R. Polymer-Based Drug Delivery to the Brain. Sci. Med. 1996, 3, 52−61. (78) Langer, R.; Peppas, N. A. Advances in Biomaterials, Drug Delivery, and Bionanotechnology. AIChE J. 2003, 49, 2990−3006. (79) Fleming, A. B.; Saltzman, W. M. Pharmacokinetics of the Carmustine Implant. Clin. Pharmacokinet. 2002, 41, 403−419. (80) Domb, A. J.; Israel, Z. H.; Elmalak, O.; Teomim, D.; Bentolila, A. Preparation and Characterization of Carmustine Loaded Polyanhydride Wafers for Treating Brain Tumors. Pharm. Res. 1999, 16, 762− 765. (81) Langer, R. Polymer Implants for Drug Delivery in the Brain. J. Controlled Release 1991, 16, 53−59. (82) Fu, Y.; Kao, W. J. Drug Release Kinetics and Transport Mechanisms of Non-Degradable and Degradable Polymeric Delivery Systems. Expert Opin. Drug Delivery 2010, 7, 429−444. (83) Van De Wetering, P.; Metters, A. T.; Schoenmakers, R. G.; Hubbell, J. A. Poly (Ethylene Glycol) Hydrogels Formed by Conjugate Addition with Controllable Swelling, Degradation, and Release of Pharmaceutically Active Proteins. J. Controlled Release 2005, 102, 619− 627. (84) Ito, T.; Fraser, I. P.; Yeo, Y.; Highley, C. B.; Bellas, E.; Kohane, D. S. Anti-Inflammatory Function of an In Situ Cross-Linkable Conjugate Hydrogel of Hyaluronic Acid and Dexamethasone. Biomaterials 2007, 28, 1778−1786. (85) Mandal, T. K.; Bostanian, L. A.; Graves, R. A.; Chapman, S. R. Poly (D, L-Lactide-Co-Glycolide) Encapsulated Poly (Vinyl Alcohol) Hydrogel as a Drug Delivery System. Pharm. Res. 2002, 19, 1713−1719. (86) Rezvantalab, S.; Drude, N. I.; Moraveji, M. K.; Güvener, N.; Koons, E. K.; Shi, Y.; Lammers, T.; Kiessling, F. PLGA-Based Nanoparticles in Cancer Treatment. Front. Pharmacol. 2018, 9, 01260. (87) Zhang, W.; Xu, W.; Ning, C.; Li, M.; Zhao, G.; Jiang, W.; Ding, J.; Chen, X. Long-Acting Hydrogel/Microsphere Composite Sequentially Releases Dexmedetomidine and Bupivacaine for Prolonged Synergistic Analgesia. Biomaterials 2018, 181, 378−391. (88) Pek, Y. S.; Pitukmanorom, P.; Ying, J. Y. Sustained Release of Bupivacaine for Post-Surgical Pain Relief Using Core−Shell Microspheres. J. Mater. Chem. B 2014, 2, 8194−8200. (89) Weaver, B. A. How Taxol/Paclitaxel Kills Cancer Cells. Mol. Biol. Cell 2014, 25, 2677−2681. (90) Dubikovskaya, E. A.; Thorne, S. H.; Pillow, T. H.; Contag, C. H.; Wender, P. A. Overcoming Multidrug Resistance of Small-Molecule Therapeutics through Conjugation with Releasable Octaarginine Transporters. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 12128−12133. (91) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science 2010, 328, 1031−1035. (92) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-Assembly and Mineralization of Peptide-Amphiphile Nanofibers. Science 2001, 294, 1684−1688. (93) Palmer, L. C.; Stupp, S. I. Molecular Self-Assembly into OneDimensional Nanostructures. Acc. Chem. Res. 2008, 41, 1674−1684.
(57) Lin, R.; Cheetham, A. G.; Zhang, P.; Lin, Y.-A.; Cui, H. Supramolecular Filaments Containing a Fixed 41% Paclitaxel Loading. Chem. Commun. 2013, 49, 4968−4970. (58) Zhang, P.; Cheetham, A. G.; Lock, L. L.; Cui, H. Cellular Uptake and Cytotoxicity of Drug-Peptide Conjugates Regulated by Conjugation Site. Bioconjugate Chem. 2013, 24, 604. (59) Chen, Z.; Xing, L.; Fan, Q.; Cheetham, A. G.; Lin, R.; Holt, B.; Chen, L.; Xiao, Y.; Cui, H. Drug-Bearing Supramolecular Filament Hydrogels as Anti-Inflammatory Agents. Theranostics 2017, 7, 2003. (60) Wang, Y.; Cheetham, A. G.; Angacian, G.; Su, H.; Xie, L.; Cui, H. Peptide-Drug Conjugates as Effective Prodrug Strategies for Targeted Delivery. Adv. Drug Delivery Rev. 2017, 110-111, 112−126. (61) Li, Y.; Shawgo, R. S.; Tyler, B.; Henderson, P. T.; Vogel, J. S.; Rosenberg, A.; Storm, P. B.; Langer, R.; Brem, H.; Cima, M. J. In Vivo Release from a Drug Delivery Mems Device. J. Controlled Release 2004, 100, 211−219. (62) Baltes, S.; Freund, I.; Lewis, A. L.; Nolte, I.; Brinker, T. Doxorubicin and Irinotecan Drug-Eluting Beads for Treatment of Glioma: A Pilot Study in a Rat Model. J. Mater. Sci.: Mater. Med. 2010, 21, 1393−1402. (63) Scott, A. W.; Tyler, B. M.; Masi, B. C.; Upadhyay, U. M.; Patta, Y. R.; Grossman, R.; Basaldella, L.; Langer, R. S.; Brem, H.; Cima, M. J. Intracranial Microcapsule Drug Delivery Device for the Treatment of an Experimental Gliosarcoma Model. Biomaterials 2011, 32, 2532− 2539. (64) Nance, E.; Zhang, C.; Shih, T.-Y.; Xu, Q.; Schuster, B. S.; Hanes, J. Brain-Penetrating Nanoparticles Improve Paclitaxel Efficacy in Malignant Glioma Following Local Administration. ACS Nano 2014, 8, 10655−10664. (65) Miladi, I.; Aloy, M.-T.; Armandy, E.; Mowat, P.; Kryza, D.; Magné, N.; Tillement, O.; Lux, F.; Billotey, C.; Janier, M.; et al. Combining Ultrasmall Gadolinium-Based Nanoparticles with Photon Irradiation Overcomes Radioresistance of Head and Neck Squamous Cell Carcinoma. Nanomedicine 2015, 11, 247−257. (66) Guerrero-Cázares, H.; Tzeng, S. Y.; Young, N. P.; Abutaleb, A. O.; Quiñones-Hinojosa, A.; Green, J. J. Biodegradable Polymeric Nanoparticles Show High Efficacy and Specificity at DNA Delivery to Human Glioblastoma In Vitro and In Vivo. ACS Nano 2014, 8, 5141− 5153. (67) Akilo, O. D.; Choonara, Y. E.; Strydom, A. M.; du Toit, L. C.; Kumar, P.; Modi, G.; Pillay, V. An in vitro Evaluation of a CarmustineLoaded Nano-Co-Plex for Potential Magnetic-Targeted Intranasal Delivery to the Brain. Int. J. Pharm. 2016, 500, 196−209. (68) Ranganath, S. H.; Fu, Y.; Arifin, D. Y.; Kee, I.; Zheng, L.; Lee, H.S.; Chow, P. K.-H.; Wang, C.-H. The Use of Submicron/Nanoscale PLGA Implants to Deliver Paclitaxel with Enhanced Pharmacokinetics and Therapeutic Efficacy in Intracranial Glioblastoma in Mice. Biomaterials 2010, 31, 5199−5207. (69) Tyler, B.; Fowers, K. D.; Li, K. W.; Recinos, V. R.; Caplan, J. M.; Hdeib, A.; Grossman, R.; Basaldella, L.; Bekelis, K.; Pradilla, G.; et al. A Thermal Gel Depot for Local Delivery of Paclitaxel to Treat Experimental Brain Tumors in Rats: Laboratory Investigation. J. Neurosurg. 2010, 113, 210−217. (70) Matthes, K.; Mino-Kenudson, M.; Sahani, D. V.; Holalkere, N.; Fowers, K. D.; Rathi, R.; Brugge, W. R. EUS-Guided Injection of Paclitaxel (Oncogel) Provides Therapeutic Drug Concentrations in the Porcine Pancreas (with Video). Gastrointest. Endosc. 2007, 65, 448− 453. (71) Zhao, F.; Ma, M. L.; Xu, B. Molecular Hydrogels of Therapeutic Agents. Chem. Soc. Rev. 2009, 38, 883−891. (72) Sawyer, A. J.; Saucier-Sawyer, J. K.; Booth, C. J.; Liu, J.; Patel, T.; Piepmeier, J. M.; Saltzman, W. M. Convection-Enhanced Delivery of Camptothecin-Loaded Polymer Nanoparticles for Treatment of Intracranial Tumors. Drug Delivery Transl. Res. 2011, 1, 34−42. (73) Chen, P.-Y.; Ozawa, T.; Drummond, D. C.; Kalra, A.; Fitzgerald, J. B.; Kirpotin, D. B.; Wei, K.-C.; Butowski, N.; Prados, M. D.; Berger, M. S.; et al. Comparing Routes of Delivery for Nanoliposomal Irinotecan Shows Superior Anti-Tumor Activity of Local AdminJ
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
Article
ACS Nano (94) Lin, Y.-A.; Ou, Y.-C.; Cheetham, A. G.; Cui, H. Supramolecular Polymers Formed by ABC Miktoarm Star Peptides. ACS Macro Lett. 2013, 2, 1088−1094. (95) He, C.; Han, Y.; Fan, Y.; Deng, M.; Wang, Y. Self-Assembly of Aβ-Based Peptide Amphiphiles with Double Hydrophobic Chains. Langmuir 2012, 28, 3391−3396. (96) Shimada, T.; Sakamoto, N.; Motokawa, R.; Koizumi, S.; Tirrell, M. Self-Assembly Process of Peptide Amphiphile Worm-Like Micelles. J. Phys. Chem. B 2012, 116, 240−243. (97) Lock, L. L.; Reyes, C. D.; Zhang, P.; Cui, H. Tuning Cellular Uptake of Molecular Probes by Rational Design of Their Assembly into Supramolecular Nanoprobes. J. Am. Chem. Soc. 2016, 138, 3533−3540. (98) Paramonov, S. E.; Jun, H.-W.; Hartgerink, J. D. Self-Assembly of Peptide− Amphiphile Nanofibers: The Roles of Hydrogen Bonding and Amphiphilic Packing. J. Am. Chem. Soc. 2006, 128, 7291−7298. (99) Stuart, M. C.; van de Pas, J. C.; Engberts, J. B. The Use of Nile Red to Monitor the Aggregation Behavior in Ternary Surfactant− Water−Organic Solvent Systems. J. Phys. Org. Chem. 2005, 18, 929− 934. (100) Wang, K.; Zhang, X.; Liu, Y.; Liu, C.; Jiang, B.; Jiang, Y. Tumor Penetrability and Anti-Angiogenesis Using iRGD-Mediated Delivery of Doxorubicin-Polymer Conjugates. Biomaterials 2014, 35, 8735−8747.
K
DOI: 10.1021/acsnano.9b01689 ACS Nano XXXX, XXX, XXX−XXX
